Anticancer compounds

ABSTRACT

This invention features compounds having formula (I): 
                 
 
wherein, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , T, X, and Y are as defined herein. This invention also features a method for treating cancer. The method includes administrating to a subject in need thereof a compound of formula (I).

This application claims the benefit of prior U.S. prov. app. 60/417,785filed Oct. 11, 2002.

BACKGROUND

Podophyllotoxin is a naturally occurring compound extracted from amandrake plant. Some derivatives of podophyllotoxin, e.g., etoposide andteniposide, have been studied for use in chemotherapy for cancer. (See,e.g., Jardine (1980) Anticancer Agents Based on Natural Products Models;Academic Press: New York, p 319; Issell (1982) Cancer Chemother.Pharmacol. 7: 73; and Lee et al. (1995) Food and Drug Analysis. 3:209).These derivatives inhibit topoisomerase II by stabilizing atopoisomerase II-DNA complex in which the DNA is cleaved and remainscovalently linked to the enzyme. This inhibition leads to cell death.See, e.g., Osheroff et al. (1991) BioEssays 13: 269; Alton & Harris(1993) Br. J. Haematol. 85: 241-245, Cho et al. (1996) J. Med. Chem. 39:1383; MacDonald et al. (1991) DNA Topoisomerase in Cancer; OxfordUniversity Press: New York. It is known that the aforementionedpodophyllotoxin derivatives have several limitations such as developmentof drug resistance, myelo-suppression, and poor oral bioavailability.Thus, identification of novel compounds that also target topoisomeraseII can lead to new therapeutics for treating or preventing cancer orsymptoms associated with cancer.

SUMMARY

The present invention is based, in part, on the discovery of novelpodophyllotoxin derivatives that possess anticancer activities.

In one aspect, this invention features compounds having formula (I) thatincludes a tetracyclic-fused ring:

each of R₁, R₂, R₃ and R₇ independently is H or alkyl; each of R₄ and R₆independently is alkyl; R₅ is H or P(O)(OR_(a))₂, in which R_(a) is H oralkyl; T is H, or together with X is ═N; X is a bond, O, S, or NR_(b),in which R_(b) is H or alkyl; or together with T, is ═N; and Y is5-membered heteroaryl or heterocyclyl, each of which optionallysubstituted with one or more of halogen, alkyl, cyclyl, aryl,heteroaryl, heterocyclyl, —OR_(c), —NR_(c)R_(c)′, —SR_(c), —CN, —NO₂,—SO₂R_(c), —C(O)OR_(c), —C(O)NR_(c)R_(c)′, —NHC(O)R_(c),—(CH₂)_(q)OPO₃H₂, —CH₂C(O)NOR_(c)″, and

in which each of R_(c) and R_(c)′ independently is H or alkyl; R_(c)″ isH, alkyl, or silyl; Z is O or NH; each of m and n independently is 0 or1; p is 0, 1, or 2; q is 1, 2, 3, or 4; and each of R₈ and R₉independently is H, alkyl, aryl, heteroaryl, heterocyclyl, —OR_(d),—NR_(d)R_(d)′, —SR_(d), —CN, —NO₂, —SO₂R_(d)—C(O)OR_(d),—C(O)NR_(d)R_(d)′, —NHC(O)R_(d), or —NHC(O)OR_(d), in which each ofR_(d) and R_(d)′ independently is H or alkyl.

Referring to the just-described compounds, for a subset of thesecompounds X is NH and T is H. Another subset of the compounds are thosewherein each of R₁, R₂, R₃, and R₇ is H; or each of R₄ and R₆ is methyl;or R₅ is H.

Further, another subset of the compounds are those wherein Y isheteroaryl substituted with

In some embodiments, m is 1. In these compounds, the heteroaryl can be

X can be NH; T can be H; each of R₁, R₂, R₃, and R₇ can be H; each of R₄and R₆ can be methyl; and R₅ can be H. In other embodiments, m is 0.

Unless specifically pointed out, alkyl, alkenyl, aryl, heteroaryl,cyclyl, and heterocyclyl mentioned herein include both substituted andunsubstituted moieties. The term “substituted” refers to one or moresubstituents (which may be the same or different), each replacing ahydrogen atom. Examples of substituents include halogen, cyano, nitro,hydroxyl, amino, mercapto, alkyl, alkenyl, alkynyl, aryl, heteroaryl,cyclyl, heterocyclyl, alkyloxy, aryloxy, alksulfanyl, arylsulfanyl,alkylamino, arylamino, dialkylamino, diarylamino, alkylcarbonyl,arylcarbonyl, heteroarylcarbonyl, alkylcarboxyl, arylcarboxyl,heteroarylcarboxyl, alkyloxycarbonyl, aryloxycarbonyl,heteroaryloxycarbonyl, alkylcarbamido, arylcarbamido, heterocarbamido,alkylcarbamyl, arylcarbamyl, heterocarbamyl, wherein each of alkyl,alkenyl, aryl, heteroaryl, cyclyl, and heterocyclyl is optionallysubstituted with halogen, cyano, nitro, hydroxyl, amino, mercapto,alkyl, aryl, heteroaryl, alkyloxy, aryloxy, alkylcarbonyl, arylcarbonyl,alkylcarboxyl, arylcarboxyl, alkyloxycarbonyl, or aryloxycarbonyl.

As used herein, the term “alkyl” refers to a straight-chained orbranched alkyl group containing 1 to 6 carbon atoms. Examples of alkylgroups include methyl, ethyl, n-propyl, isopropyl, tert-butyl, andn-pentyl.

The term “alkenyl” refers to a straight-chained or branched alkenylgroup containing 2 to 6 carbon atoms. Examples of alkenyl groups includevinyl, allyl (2-propenyl), dimethylallyl, and butenyl.

The term “aryl” refers to a hydrocarbon ring system (monocyclic totricyclic) having at least one aromatic ring. Examples of aryl groupsinclude, but are not limited to, phenyl, naphthyl, and anthracenyl.

The term “heteroaryl” refers to a hydrocarbon ring system (monocyclic totricyclic) having at least one aromatic ring which contains at least oneheteroatom (e.g., O, N, or S) as part of the ring in place of carbonatoms. Examples of heteroaryl groups include, but are not limited to,furyl, pyrrolyl, pyrazolyl, thiophenyl, thiadiazolyl, tetrazolyl,triazolyl, triazinyl, thienyl, oxazolyl, isoxazolyl, imidazolyl,thiazolyl, isothiazolyl, benzimidazolyl, pyridinyl, pyrimidinyl,quinazolinyl, indolyl, indiazolyl, isoindolyl, benzotriazolyl, purinyl,benzothiazolyl, benzoisothiazolyl, and benzothiadiazolyl.

The term “5-membered heteroaryl” refers to a ring system (monocyclic totricyclic) containing at least one aromatic ring which has 5 ring atomsincluding one or more heteroatoms (e.g., O, N, or S). Examples of5-membered heteroaryl include, but are not limited to, furyl, pyrrolyl,pyrazolyl, thiadiazolyl, tetrazolyl, triazolyl, thienyl, oxazolyl,isoxazolyl, imidazolyl, thiazolyl, isothiazolyl, benzimidazolyl,benzotriazolyl, purinyl, benzothiazolyl, benzoisothiazolyl, andbenzothiadiazolyl.

The term “cyclyl” refers to a hydrocarbon ring system containing 3 to 8carbon ring members. It includes saturated and unsaturated cycles.Examples of cyclyl groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, methylcyclohexyl, and cycloheptyl.

The term “heterocyclyl” refers to a hydrocarbon ring system containing 3to 8 ring members that have at least one heteroatom (e.g., N, O, or S)as part of the ring in place of carbon atoms. It includes saturated andunsaturated heterocycles. Examples of heterocyclyl groups include, butare not limited to, piperidyl, morpholinyl, pyranyl, dioxanyl, andpiperazinyl.

Set forth below are exemplary compounds of this invention.

Compounds 1-210 having the following formula:

Compound ID R X T R′  1

NH H H  2

NH H H  3

NH H H  4

NH H H  5

NH H H  6

NH H H  7

NH H H  8

NH H H  9

NH H H  10

NH H H  11

NH H H  12

NH H H  13

NH H H  14

NH H H  15

NH H H  16

NH H H  17

NH H H  18

NH H H  18a

NH H H  19

NH H H  20

NH H H  21

NH H H  22

NH H H  23

NH H H  24

NH H H  25

NH H H  26

NH H H  27

NH H H  28

NH H H  29

NH H H  29a

NH H H  30

NH H H  31

NH H H  32

NH H H  33

NH H H  34

NH H H  35

NH H H  36

NH H H  37

NH H H  38

NH H H  39

NH H H  40

NH H H  41

NH H H  42

NH H H  43

NH H H  44

NH H H  45

NH H H  46

NH H H  47

NH H H  48

NH H H  49

NH H H  50

NH H H  51

NH H H  52

NH H H  53

NH H H  54

NH H H  55

NH H H  56

NH H H  57

NH H H  58

NH H H  59

NH H H  60

NH H H  61

NH H H  62

S H H  63

NH H H  64

NH H H  65

NH H H  66

NH H H  67

NH H H  68

NH H H  69

NH H H  70

NH H H  71

S H H  72

NH H H  73

NH H H  74

NH H H  75

NH H H  76

NH H H  77

NH H H  78

NH H H  79

NH H H  80

NH H H  81

NH H H  82

NH H H  83

NH H H  84

NH H H  85

NH H H  86

NH H H  87

NH H H  88

O H H  89

NH H H  90

O H H  91

NH H H  92

═N H  93

NH H H  94

NH H H  95

NH H H  96

NH H H  97

NH H H  98

NH H H  99

NH H H 100

NH H H 101

NH H H 102

NH H H 103

NH H H 104

NH H H 105

NH H H 106

NH H H 107

NH H H 108

NH H H 109

NH H H 110

NH H H 111

NH H H 112

NH H H 113

NH H H 114

NH H H 115

NH H H 116

NH H H 117

NH H H 118

NH H H 119

NH H H 120

NH H H 121

NH H H 122

NH H H 123

NH H H 124

NH H H 125

NH H H 126

NH H H 127

NH H H 128

NH H H 129

NH H H 130

NH H H 131

NH H H 132

NH H H 133

NH H H 134

NH H H 135

NH H H 136

NH H H 137

NH H H 138

NH H H 139

NH H H 140

NH H H 141

NH H H 142

NH H H 143

NH H H 144

NH H H 145

NH H H 146

NH H H 147

NH H H 148

NH H H 149

NH H H 150

NH H H 151

NH H H 152

NH H H 153

S H H 154

NH H H 155

NH H H 156

NH H H 157

NH H H 158

NH H H 159

NH H H 160

O H H 161

NH H H 162

— H H 163

NH H H 164

NH H H 165

NH H H 166

S H H 167

NH H H 168

NH H H 169

NH H H 170

NH H H 171

O H H 172

NH H H 173

NH H H 174

NH H H 175

NH H H 176

— H H 177

— H H 178

NH H H 179

NH H H 180

NH H H 181

NH H H 182

NH H H 183

NH H H 184

NH H H 185

NH H H 186

NH H H 187

NH H H 188

NH H H 189

NH H H 190

NH H H 191

NH H H 192

NH H H 193

NH H H 194

NH H H 195

NH H H 196

NH H H 197

NH H H 198

NH H H 199

NH H H 200

NH H H 201

NH H H 202

NH H H 203

NH H H 204

NH H H 205

NH H H 206

NH H H 207

NH H OPO₃H₂ 208

NH H OPO₃H₂ 209

NH H OPO₃H₂ 210

NH H OPO₃H₂

The podophyllotoxin derivatives described above include the compoundsthemselves, as well as their salts and their prodrugs, if applicable.The salts, for example, can be formed between a positively chargedsubstituent (e.g., amino) on a compound and an anion. Suitable anionsinclude, but are not limited to, chloride, bromide, iodide, sulfate,nitrate, phosphate, citrate, methanesulfonate, tartrate,trifluoroacetate, and acetate. Likewise, a negatively chargedsubstituent (e.g., carboxylate) on a compound can form a salt with acation. Suitable cations include, but are not limited to, sodium ion,potassium ion, magnesium ion, calcium ion, and an ammonium cation suchas teteramethylammonium ion. Examples of prodrugs include esters andother pharmaceutically acceptable derivatives, which, uponadministration to a subject, are capable of providing thepodophyllotoxin derivatives described above.

In addition, the just-described podophyllotoxin derivatives may have oneor more double bonds, or one or more additional asymmetric centers. Suchcompounds can occur as racemates, racemic mixtures, single enantiomers,individual diastereomers, and diastereomeric mixtures.

Another aspect of the present invention relates to a pharmaceuticalcomposition that contains a pharmaceutically acceptable carrier and aneffective amount of at least one of the podophyllotoxin derivativesdescribed above.

A further aspect of this invention relates to a method for treatingcancer, e.g., carcinoma or sarcoma. The method includes administering toa subject in need thereof an effective amount of one or more theaforementioned podophyllotoxin derivatives.

As used herein, “cancer” refers to a cellular tumor. Cancer cells havethe capacity for autonomous growth, i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth. The term is meant toinclude all types of cancerous growths or oncogenic processes,metastatic tissues or malignantly transformed cells, tissues, or organs,irrespective of histopathologic type, or stage of invasiveness. Examplesof cancers include, but are not limited to, carcinoma and sarcoma suchas leukemia, sarcomas, osteosarcoma, lymphomas, melanoma, ovariancancer, skin cancer, testicular cancer, gastric cancer, pancreaticcancer, renal cancer, breast cancer, prostate cancer, colorectal cancer,cancer of the head and neck, brain cancer, esophageal cancer, bladdercancer, adrenal cortical cancer, lung cancer, bronchus cancer,endometrial cancer, nasopharyngeal cancer, cervical or hepatic cancer,or cancer of unknown primary site. In addition, cancer can be associatedwith a drug resistance phenotype.

Also within the scope of this invention is a composition containing oneor more of the podophyllotoxin derivatives described above for use intreating cancer, and the use of such a composition for the manufactureof a medicament for cancer treatment.

Other features or advantages of the present invention will be apparentfrom the following detailed description of several embodiments, and alsofrom the appending claims.

DETAILED DESCRIPTION

The podophyllotoxin derivatives described above can be prepared bymethods well known in the art, as well as by the synthetic routesdisclosed herein. See, e.g., Wang et al. (1992) Yaoxue Xuebao 27: 656;Lee et al. (1989) J. Nat. Prod. 52: 606; and Chen et al. (2000) ChineseChemical Letters 11: 505. For example, as shown in the scheme below, onecan use podophyllotoxin as a starting material. Bromination ofpodophyllotoxin gives an intermediate,4′-O-demethyl-4β-bromo-4-desoxypodophylotoxin (Kuhn, et al. (1969) Helv.Chim. Acta 52: 944). The intermediate reacts with an amino substitutedheteroaryl or heterocyclyl side chain in the presence of a weak base,e.g., barium carbonate, to provide a podophyllotoxin derivative of thisinvention as shown in the scheme below (Y in the scheme is as defined inSummary). The amino substituted heteroaryl or heterocyclyl moiety issynthesized by a cyclization reaction followed by modifications on itssubstituents.

Alternatively, a compound of this invention can be synthesized bycoupling of the aforementioned intermediate with a mercapto or hydroxylsubstituted heteroaryl.

The chemicals used in the above-described synthetic route may include,for example, solvents, reagents, catalysts, protecting group anddeprotecting group reagents. The methods described above may alsoadditionally include steps, either before or after the steps describedspecifically herein, to add or remove suitable protecting groups inorder to ultimately allow synthesis of the podophyllotoxin derivative.In addition, various synthetic steps may be performed in an alternatesequence or order to give the desired compounds. Synthetic chemistrytransformations and protecting group methodologies (protection anddeprotection) useful in synthesizing applicable podophyllotoxinderivatives are known in the art and include, for example, thosedescribed in R. Larock, Comprehensive Organic Transformations, VCHPublishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups inOrganic Synthesis, 3^(rd) Ed., John Wiley and Sons (1999); L. Fieser andM. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, JohnWiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagentsfor Organic Synthesis, John Wiley and Sons (1995) and subsequenteditions thereof.

A podophyllotoxin derivative thus synthesized can be further purified bya method such as column chromatography, High-Performance LiquidChromatography (HPLC), High-Performance Flash Chromatography (HPFC), orrecrystallization.

Podophyllotoxin derivative phosphate prodrugs of this invention arefurther prepared according to the method described in U.S. Pat. No.4,904,768 and U.S. Pat. No. 5,606,039. They are synthesized by reactingpodophyllotoxin derivatives with phosphorous oxychloride in anappropriate solvent, e.g., acetonitrile, in the presence of an organicbase, e.g., N,N-diisopropylethtylamine.

This invention features a method for treating cancer. The methodincludes administering to a subject in need thereof an effective amountof one or more podophyllotoxin derivatives described in Summary and apharmaceutically acceptable carrier. The term “treating” is defined asthe application or administration of a composition including thepodophyllotoxin derivative to a subject, who has cancer, a symptom ofcancer, or a predisposition toward cancer, with the purpose to cure,heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affectcancer, the symptoms of cancer, or the predisposition toward cancer. “Aneffective amount” is defined as the amount of a podophyllotoxin compoundwhich, upon administration to a subject in need thereof, is required toconfer therapeutic effect on the subject. An effective amount of apodophyllotoxin derivative may range from about 0.2 mg/Kg to about 60mg/Kg. Effective doses also vary, as recognized by those skilled in theart, depending on route of administration, excipient usage, and thepossibility of co-usage with other therapeutic treatments such as use ofother anticancer agents or radiation therapy. Examples of the otheranticancer agents include paclitaxel, docitaxel, doxorubicin,daunorubicin, epirubicin, fluorouracil, melphalan, cis-platin,carboplatin, cyclophosphamide, mitomycin C, methotrexate, mitoxantrone,vinblastine, vincristine, ifosfamide, teniposide, etoposide, bleomycin,leucovorin, cytarabine, dactinomycin, interferon alpha, streptozocin,prednisolone, procarbazine, irinotecan, topotecan, colony stimulatingfactor, granulocyte/monocyte colony stimulating factor, and imatinibmesylate.

To practice the method of the present invention, a podophyllotoxinderivative can be administered orally, parenterally, by inhalationspray, or via an implanted reservoir. The term “parenteral” as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques.

A composition for oral administration can be any orally acceptabledosage form including, but not limited to, tablets, capsules, emulsionsand aqueous suspensions, dispersions and solutions. Commonly usedcarriers for tablets include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added to tablets.For oral administration in a capsule form, useful diluents includelactose and dried corn starch. When aqueous suspensions or emulsions areadministered orally, the active ingredient can be suspended or dissolvedin an oily phase combined with emulsifying or suspending agents. Ifdesired, certain sweetening, flavoring, or coloring agents can be added.

A sterile injectable composition (e.g., aqueous or oleaginoussuspension) can be formulated according to techniques known in the artusing suitable dispersing or wetting agents (such as, for example, Tween80) and suspending agents. The sterile injectable preparation can alsobe a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example, as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that canbe employed are mannitol, water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium (e.g., synthetic mono- ordi-glycerides). Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions can also contain a long-chain alcohol diluent or dispersant,or carboxymethyl cellulose or similar dispersing agents.

An inhalation composition can be prepared according to techniqueswell-known in the art of pharmaceutical formulation and can be preparedas solutions in saline, employing benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

A carrier in a pharmaceutical composition must be “acceptable” in thesense of being compatible with the active ingredient of the formulation(and preferably, capable of stabilizing it) and not deleterious to thesubject to be treated. For example, solubilizing agents, such ascyclodextrins (which form specific, more soluble complexes withpodophyllotoxin derivatives), can be utilized as pharmaceuticalexcipients for delivery of podophyllotoxin derivatives. Examples ofother carriers include colloidal silicon dioxide, magnesium stearate,cellulose, sodium lauryl sulfate, and D&C Yellow #10.

Podophyllotoxin derivatives of this invention can be preliminarilyscreened for their efficacy in treating cancer by in vitro assays. Forexample, podophyllotoxin derivatives can be tested for theircytotoxicity against KB cells (nasopharyngeal carcinoma). Morespecifically, a test compound can be added to a culture of KB cells andits IC₅₀ (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of cell growth) is determined using thesulforhodamine B (a protein binding dye) assay as described in J.N.C.I.(1990) 82: 1107. The podophyllotoxin derivatives of this invention arealso tested for their abilitives to inhibit DNA topoisomerase II invitro as described in Cho et al. (1996 J. Med. Chem. 39: 1396) and tostimulate protein-linked DNA breaks (PLDB) in KB cells as described inRowe et al. (1986 Cancer Res. 46:2021). DNA topoisomerase II is a wellknown target for cancer treatment drugs. See, e.g., MacDonald et al.(1991) DNA Topoisomerase in Cancer; Oxford University Press: New York.

Podophyllotoxin derivatives of this invention can further be screenedfor their efficacy in treating caner by in vivo assays. For example, atest compound can be injected into an animal (e.g., a mouse model) andits therapeutic effects are then accessed. Based on the results, anappropriate dosage range and administration route can also bedetermined.

Without further elaboration, it is believed that the above descriptionhas adequately enabled the present invention. The following specificembodiments are, therefore, to be construed as merely illustrative, andnot limitative of the remainder of the disclosure in any way whatsoever.All of the publications cited herein are hereby incorporated byreference in their entirety.

Chemical Syntheses

As used herein, melting points were determined on a Fisher-John meltingpoint apparatus and are uncorrected. Proton Nuclear Magnetic Resonance(¹H NMR) spectra were measured on a Varian 300 or a Bruker 400 (ifindicated) spectrometer with tetramethylsilane (TMS) as the internalstandard. Chemical shifts are reported in δ (ppm). Mass spectra (MS)were obtained on an API 3000 LC/MS/MS spectrometer. Flash columnchromatography was performed on silica gel (100-200 mesh). HPFC wasconducted on a Biotage Horizon system. Precoated silica gel plates(Kieselgel 60 F₂₅₄, 0.25 mm) were used for thin layer chromatography(TLC) analysis.

Synthesis of Compounds 1-2, 7-9, 12-13, 15-18, 20-26, 29, 29a, 31, and32

These compounds were synthesized starting from podophyllotoxin as shownin Scheme 1 below. Bromination of podophyllotoxin gave an intermediate,4′-O-demethyl-4β-bromo-4-desoxypodophylotoxin (referred to as DBDhereinafter). Hydrolyzing the methyl ester in Compound 7 with 2N HCl inTHF afforded Compound 9. Removal of the tert-butyl and Boc groups inCompounds 22, 25 and 29 with trifluoroacetic acid (TFA), in the presenceof anisole, gave Compounds 20, 23 and 29a, respectively. Morespecifically, 4β-N-linked -(substituted heteroaryl)-4′-O-demethyl4-epipodophyllotoxin was synthesized as follows. To a solution of DBD inan appropriate solvent mixture (e.g., THF and 1,2-dichloroethane (DCE)(1:1)/or acetonitrile (1:1)) was added an amino substituted heteroaryl(1.2 equivalent) and BaCO₃ (1.5 equivalents). The mixture was heated toreflux under nitrogen with TLC or LC-MS monitoring. The reaction mixturewas cooled to room temperature and a solid was formed and filtered. Thefiltrate thus obtained was concentrated to provide a crude product. Thecrude product was purified by silica gel column chromatography withCH₂Cl₂:EtOAc:acetone, EtOAc:hexanes:MeOH, or CH₂Cl₂:MeOH as the eluant.

Analytical data on two compounds are shown below.

Compound 2, i.e., 4′-O-demethyl-4β-[4″-(ethylL-tryptophan-N-acetyl)-2″-thiazolyl amino]-4-desoxypodophyllotoxin. ESIMS: 754 [M+H], 753 [M−H]; ¹H NMR (300 MHz, CDCl₃) δ: 8.35 (br s, 1H,NH), 7.96 (d, J=8 Hz, 1H, 7′″-H), 7.42 (d, J=8 Hz, 1H, 4′″-H), 7.05 (s,1H, 3′″-H), 6.97 (m, 2H, 5′″, 6′″-H), 6.82 (s, 1H, H-5), 6.49 (s, 1H,8-H), 6.30 (s, 2H, 2′, 6′-H), 6.22 (s, 1H, 5″-H), 5.95 (2H, d, J=12 Hz,OCH₂O), 4.98 (m, 2H, 4, 9′″-H), 4.51 (d, J=5 Hz, 1H, 1-H), 4.12 (t, J=7Hz, 2H, OCH ₂CH₃), 3.82 (s, 6H, 3′, 5′-OCH₃), 3.52 (d, J=7 Hz, 2H,6″-H), 3.42 (m, 2H, 11, 8′″-H), 3.28 (dd, J=4, 15 Hz, 1H, 8′″-H), 3.16(t, J=10 Hz, 1H, 11-H), 2.78 (dd, J=5, 14 Hz, 1H, 2-H), 2.58 (m, 1H,3H).

Compound 32, i.e., 4′-O-demethyl-4β-[4″-(ethylL-phenylglycyl-N-acetyl)-2″-thiazolyl amino]-4-desoxypodophyllotoxin.Amorphous, mp 150-153° C. (dec.); ESI MS: 700.4 [M−H]; ¹H NMR (300 MHz,CDCl₃): δ 8.21 (br. d, J=7.1 Hz, 1H, NH of amino acid), 7.35-7.23 (m,5H, Benzene ring of amino acid), 6.90 (s, 1H, 5-H), 6.52 (s, 1H, 8-H),6.32 (s, 2H, 2′-H, 6′-H), 6.29 (s, 1H, S—CH—), 5.96 (d, 2H, J=11.0 Hz,—OCH₂O—), 5.54 (d, J=7.1 H, 1H, CONH—CH), 5.32 (br. s, 1H, 4-H), 4.57(d, J=4.4 Hz, 1H, 1-H), 4.25 (dd, J=6.6, 8.8 Hz, 1H, 11β-H), 4.14 (q,J=7.1 Hz, 2H, CH ² CH₃), 3.92 (t, J=9.6 Hz, 1H, 11α-H), 3.77 (s, 6H,3′,5′-OCH₃), 3.52 (s, 2H, CH ² CONH), 2.98 (m, 2H, 2-H, 3-H), 1.26, 1.18(total 3H, both t, J=7.1 Hz, CH₂ CH ³ , isomer ratio=2:5).

Synthesis of Compounds 6, 11, 36-42, 77-82, 96, 118, 126, 128, 130, 131,140, 145, 146, 163, and 165

Each of Compounds 36 and 37 was synthesized as follows. Reaction of anamino substituted heteroaryl with (trimethylsilyl) diazomethane (2.0 Msolution in hexanes) in a solvent of methanol and benzene yielded anintermediate. Substitution of the intermediate at C-4 position of DBDgave the desired product. See Scheme 2 below.

Analytical data on Compound 36 are shown below.

Compound 36, i.e.,4′-O-demethyl-4β-[4″-(methyl-O-acetyl)-2″-thiazolylamino]-4-desoxypodophyllotoxin.Yield 59%; Amorphous, mp 116-120° C. (dec.); ESI MS: 553 [M−H], 577[M+Na]. ¹H NMR (CDCl₃) δ 6.85 (s, 1H, 5-H), 6.52 (s, 1H, 8-H), 6.38 (s,1H, 5″-H), 6.30 (s, 2H, 2′,6′-H), 5.98 and 5.96 (dd, 2H, —OCH ² O—),5.16 (br, 1H, 4-H), 4.59 (br, 1H, 1-H), 4.40 (t, 1H, 11-H), 3.95 (t, 1H,11-H), 3.79 (s, 6H, 3′,5′-OCH ³ ), 3.73 (s, 3H, —COOCH ³ ), 3.60 (s, 2H,—CH ² COOCH₃), 3.00 (m, 2H, 2-H, 2-H).

Compounds 6, 11, 38-42, 77-82, 84, 96, 118, 126, 128, 130-131, 140,145-146, 163, and 165 were synthesized by coupling an appropriatealcohol or amine to an amino substituted heteroaryl followed byconjugation with DBD.

Analytical data on a number of compounds are shown below.

Compound 39, i.e.,4′-O-demethyl-4β-[5″-(ethoxycarbonyl)-2″-pyridylamino)]-4-desoxypodophyllotoxin.Yield 35%; Amorphous, mp 164-168° C. (dec.); ESI MS: 547 [M−H],571[M+Na]. ¹H NMR (CDCl₃) δ 8.78 (d, 1H, J=2.2 Hz, 6″-H), 8.04 and8.01(dd, J=2.2 Hz, 1H, 4″-H), 6.79 (s, 1H, 5-H), 6.55 (s, 1H, 8-H), 6.42(d, J=8.8 Hz, 1H, 5″-H), 6.33 (s, 2H, 2′,6′-H), 5.99, 5.96 (dd, J=1.6Hz, 2H, —OCH ² O—), 5.46 (br, 2H, 4-H, NH), 4.88 (d, J=5.5 Hz, 1H, 1-H),4.62 (br, 1H, 11-H), 4.40 (br, 1H, 11-H), 4.36 (q, J=7.1 Hz, 2H, CH ²CH₃), 3.79 (s, 6H, 3′,5′-OCH ³ ), 3.03 (m, 2H, 2-H, 2-H), 1.36 (t, J=7.1Hz, 3H, CH₂ CH ³ ).

Compound 84, i.e.,4′-O-demethyl-4β-[4″-(ethoxycarbonyl)-3″-pyrazolylamino]-4-desoxypodophyllotoxin.Yield 40%; White solid, mp 152-155° C. (dec.); ESI MS: 536 [M−H]. ¹H NMR(CDCl₃) δ 7.32 (s, 1H, 5″-H), 6.66 (s, 1H, 5-H), 6.62 (s, 1H, 8-H), 6.31(s, 2H, 2′,6′-H), 6.01, 6.00 (dd, J=1.1 Hz, 2H, —OCH ² O—), 5.45 (s, 1H,NH), 5.43 (d, J=4.9 Hz, 1H, 4-H), 4.70 (d, J=4.9 Hz, 1H, 1-H), 4.68 (br,1H, 11-H), 4.36 (br, 1H, 11-H), 4.25 (m, 2H, CH ² CH₃), 3.79 (s, 6H,3′,5′-OCH ³ ), 3.55 (m, 1H, 3-H,), 3.25 (dd, J=4.9 Hz, 1H, 2-H,), 1.35(t, J=7.1 Hz, 3H, CH₂ CH ³ ).

Compound 140, i.e.,4′-O-demethyl-4β-[2″-(3″-(2′″-chloro-4′″-pyridinylamino-carbonyl))-pyridinlylamino]-4-desoxypodophyllotoxin.Amorphous, mp >240° C. (dec); ESI MS: 630.0 (M−1); ¹H NMR δ (400 MHz,CDCl₃): 8.65 (1H, d, J=2.3 Hz, 3′″-H of second pyridine), 8.32 (1H, d,J=5.5 Hz, 6′″-H of second pyridine), 7.95 (1H, dd, J=2.3, 9.0 Hz, 4″-Hof first pyridine), 7.76 (1H, d, J=2.0 Hz, 6″-H of first pyridine), 7.48(1H, dd, J=2.0, 5.9 Hz, 5′″-H of second pyridine), 6.79 (1H, s, 5-H),6.56 (1H, s, 8-H), 6.50 (1H, d, J=9.0 Hz, 3″-H of first pyridine), 6.34(2H, s, 2′-H, 6′-H), 5.98 (2H, d d, J=1.2, 6.7 Hz, —OCH₂O—), 5.44 (1H,d. J=5.5 Hz, 4-H), 4.63 (1H, d, J=3.9 Hz, 1-H), 4.42 (1H, dd, J=7.0, 9.4Hz, 11α-H), 3.80 (1H, d, J=2.2, 8.8 Hz, 2-H), 3.80 (6H, s, 3′,5′-OCH₃),3.50 (1H, m, 3-H), 3.03 (1H, br. d, J=4.7 Hz, 11β-H).

Synthesis of Compound 3-5, 10, 14, 19, and 30

These compounds were synthesized as shown in Scheme 3. Analytical dataon Compound 14 are shown below.

Compound 14, i.e., 4′-O-demethyl-4β-[4″-(ethylL-phenylglycyl-N-acetyl)-2″-thiazolyl amino]-4-desoxypodophyllotoxin.Amorphous, mp 233-236° C. (dec.); ESI MS: 694.4 [M−1]; ¹H NMR (300 MHz,CDCl₃): δ 8.48 (d, J=3.3 Hz, 1H, 6″-H), 7.77 (dd, J=2.7, 8.8 Hz, 1H,4″-H), 7.34-7.21 (m, 3H, 3′″-H, 4′″-H, 5′″-H), 7.17 (dd, J=1.6, 6,6 Hz,2H, 2′″-H, 6′″-H), 6.78 (s, 1H, 5-H), 6.52 (s, 1H, 8-H), 6,46 (d, J=8.8Hz, 1H, 3″-H), 6.32 (s, 2H, 2′-H, 6′-H), 5.95 (dd, J=1.6, 6.6 Hz, 2H,—OCH₂O—), 5.00 (t, J=6.3 Hz, 1H, 4-H), 4.58 (d, J=4.9 Hz, 1H, 1-H), 4.41(br.d, J=7.1 Hz, 1H, 11β-H), 4.21 (q, J=7.4 Hz, 2H, —CH ² CH₃), 3.83(dd-like, 1H, 11α-H), 3.77 (s, 6H, 3′,5′-OCH₃), 3.22 (m, 2H, 2-H, 3-H),3.20 (m, 2H, CH ² of benzyl), 1.27 (t, J=7.1 Hz, 3H, —CH₂CH ³ ).

Synthesis of Compounds 27, 28, and 33-35

Compounds 27 and 28 were synthesized as shown in Scheme 4. Reaction of1-(3-aminopropyl)-imidazole or 4-(3-aminopropyl)-morpholine with anamino substituted heteroaryl in the presence of isobutyl chloroformateand N-methyl morpholine afforded an amide compound. The amide compoundsfurther reacted with DBD to give the desire product.

Analytical data on Compound 28 are shown below.

Compound 28:4′-O-demethyl-4β-[4″-(4′″-(3′″-aminopropyl)-morpholine-N-acetyl)-2″-thiazolylamino]-4-desoxypodophyllotoxin.Amorphous, mp 105-109° C. (dec.); ESI MS: 667 [M+H]. ¹H NMR (CDCl₃) δ6.84 (s, 1H, 5-H), 6.54 (s, 1H, 8-H), 6.35 (s, 1H, 5″-H), 6.31 (s, 2H,2′,6′-H), 6.00 (m, 2H, —OCH ² O—), 5.08 (m, 1H, 4-H), 4.60 (d, J=2.7 Hz,1H, 1-H), 4.25 (m, 2H, CH ² CH₃), 4.38 (m, 1H, 11-H), 3.88 (m, 1H,11-H), 3.79 (s, 6H, 3′,5′-OCH ³ ), 3.66 (t, 4H, 2′″,6′″-CH ² — onmorpholine ring), 3.47 (s, 2H, CH ² CO on thiazole), 3.33 (m, 2H, CONHCH² CH₂CH₂—), 3.02 (br, 2H, 2-H, 3-H,), 2.38 (m, 6H, 3′″,5′″-CH ² — onmorpholine ring and CONHCH₂CH₂CH ² —), 1.70 (m, 2H, CONHCH₂CH ² CH₂—).

Compound 33 was synthesized from a dipeptide as shown in Scheme 4.Methylation of the dipeptide under refluxing in acetyl chloride andmethanol produced a methyl ester. Reaction of the methyl ester with anamino substituted heteroaryl gave an amine compound as a white crystal.The amine compound further react with DBD to give the desired product.

Compounds 34 and 35 were synthesized by coupling of tert-butyldiphenylsilyl protected hydroxylamine to an amino substituted heteroarylfollowed by conjugation with DBD (Scheme 4). Compounds 34 and 35 wereobtained by silica gel column chromatography.

Analytical data on these two compounds are shown below.

Compound 34, i.e., 4′-O-demethyl-4β-[4″-(O-tert-butyldiphenylsilylhydroxyl)-N-acetyl)-2″-thiazolylamino]-4-desoxypodophyllotoxin. Amorphous, mp 155-158° C.(dec.); ESI MS: 792 [M−H]. ¹H NMR (CDCl₃) δ 9.10 (br, 1H, NH), 7.72 (m,4H, 2′″, 6′″-H— on O-tert-butyl diphenylsilylhydroxyl), 7.34 (m, 6H,3′″, 4′″, 5′″-H— on O-tert-butyl diphenylsilylhydroxyl), 6.68 (s, 1H,5-H), 6.56 (s, 1H, 8-H), 6.32 (s, 2H, 2′,6′-H), 6.17 (s, 1H, 5″-H), 6.00(m, 2H, —OCH ² O—), 5.97 (m, 1H, 4-H), 4.70 (br, 1H, 11-H), 4.61 (d,J=4.9 Hz, 1H, 1-H), 4.08 (br, 1H, NH), 3.88 (br, 1H, 11-H), 3.80 (s, 6H,3′,5′-OCH ³ ), 3.35 (s, 2H, CH ² CO on thiazole), 3.02 (m, 1H, 3-H),2.90(m, 1H, 2-H), 1.11 (s, 9H, CH₃ on t-butyl).

Compound 35, i.e.,4′-O-demethyl-4β-[4″-(O-hydroxylamine)-N-acetyl)-2″-thiazolylamino]-4-desoxypodophyllotoxin.Amorphous, mp 165-168° C. (softened), 210-213 (dec.); ESI MS m/e: 555[M+H], 577 [M+Na]. ¹H NMR (CDCl₃) δ 6.63 (s, 1H, 5-H), 6.52 (s, 1H,8-H), 6.34 (s, 1H, 5″-H), 6.31 (s, 2H, 2′, 3′-H), 5.97 (d, J=6.0 Hz, 2H,—OCH ² O—), 4.90 (d, J=4.4 Hz, 1H, 4-H), 4.60 (d, J=4.4 Hz, 1H, 1-H),4.35 (m, 1H, 11-H), 3.84-3.90 (br, 1H, 11-H), 3.79 (s, 6H, 3′, 5′-OCH ³), 3.44 (s, 2H, CH ² CO on thiazole), 3.25 (dd, J=5.5 Hz, 1H, 2-H), 3.00(m, 1H, 3-H).

Synthesis of Compounds 43-76, 85-90, 97-101, 103-106, 108-117, 119-125,127, 129, 132-134, 136-139, 143-144, 147, 149-158, 160-162, 164,166-178, 180-188, 192-199, and 202-206

These compounds were synthesized by reacting an amino or a hydroxylsubstituted heteroaryl with DBD in the presence of barium carbonate.

Analytical data on a number of compounds are shown below.

Compound 45, i.e.,4′-O-demethyl-4β-(5″-ethoxycarbonyl-4″-methyl-2″-thiazolylamino)-4-desoxypodophyllotoxin. Amorphous, mp 257-259° C. (dec.); ESIMS: 495.2 (M−H); ¹H NMR(300 MHz, CDCl₃+CD₃OD): δ 7.37 (s, 1H, 4-H ofthiazole), 6.83 (s, 1H, 5-H), 6.53 (s, 1H, 8-H), 6.31 (s, 2H, 2′-H,6′-H), 6.01 (br. d, J=14.3 Hz, 2H, —OCH₂O—), 5.97 (s, 1H, OH), 4.59 (d,J=4.9 Hz, 1H, 4-H), 4.47 (d, J=4.4 Hz, 1H, 1-H), 4.39 (t, J=8.8 Hz, 1H,11β-H), 4.02 (t, J=7.1 Hz, 1H, 3-H), 3.78 (s, 6H, 3′,5′-OCH₃), 3.13(dd-like, J=4.9, 13.7 Hz, 1H, 11α-H), 3.03 (m, 1H, 2-H), 2.31 (s, 3H,—CH₃).

Compound 47, i.e.,4′-O-demethyl-4β-(5″-ethoxycarbonyl-4″-methyl-2″-thiazolylamino)-4-desoxypodophyllotoxin. ESI MS: 567.3 [M−H]; (300 MHz, CDCl₃) δ:6.82 (s, 1H, 5-H), 6.53 (s, 1H, 8-H), 6.30 (s, 2H, 2′, 6′-H), 5.98 (d,J=7 Hz, 2H, OCH₂O), 5.39 (m, 1H, NH), 5.13 (d, J=4 Hz, 4-H), 4.59 (d,J=4 Hz, 1H, 1-H), 4.43 (t, J=8 Hz, 1H, 11-H), 4.28 (q, J=7 Hz, 2H, OCH₂CH₃), 3.92 (m, 1H, 11-H), 3.78 (s, 6H, 3′, 5′-OCH₃), 2.98 (m, 2H, 2,3-H), 2.55 (s, 3H, 4″-CH₃), 1.34 (t, J=7 Hz, 3H, OCH₂CH ₃).

Compound 49, i.e., 4′-O-demethyl-4β-(5″-nitro-2″-thiazolylamino)-4-desoxypodophyllotoxin. Amorphous, mp 201-203° C. (dec.); ESIMS: 526.3 (M−H); ¹H NMR (300 MHz, CDCl₃): δ 8.05 (s, 1H, —H ofthiazole), 6.81 (s, 1H, 5-H), 6.56 (s, 1H, 8-H), 6.29 (s, 2H, 2′-H,6′-H), 5.99 (br. s, 2H, —OCH₂O—), 5.28 (br. s, 1H, 4-H), 4.61 (d, J=5.0Hz, 1H, 1-H), 4.44 (br. d, J=7.4, 8.8 Hz, 1H, 11β-H), 3.89 (t, J=9.8 Hz,1H, 11α-H), 3.78 (s, 6H, 3′,5′-OCH₃), 3.04 (m, 1H, 2-H), 2.95 (dd,J=4.7, 14.0 Hz, 1H, 3-H).

Compound 50, i.e.,4′-O-demethyl-4β-(5″-nitro-2″-pyridylamino)-4-desoxypodophyllotoxin.Yellow crystals, mp >250° C. (dec.); ESI MS: 520.3 (M−H); ¹H NMR (300MHz, CDCl₃+CD₃OD): δ 9.03 (br. s, 1H, 6″-H), 8.16 (d, J=9.3 Hz, 1H,4″-H), 6.80 (d, J=3.3 Hz, 1H, 5-H), 6.56 (dd, J=3.3, 8.8 Hz, 1H, 4″-H),6.55 (d, J=3.3 Hz, 1H, 8-H), 6.35 (s, 2H, 2′-H, 6′-H), 5.98 (br. t,J=4.1 Hz, 2H, —OCH₂O—), 5.62 (br. s, 1H, 4-H), 4.64 (d, J=3.8 Hz, 1H,1-H), 4.42 (br.dd, J=8.2, 1H, 14.8 Hz, 11β-H), 3.79 (s, 6H, 3′,5′-OCH₃),3.39 (d, J=3.8 Hz, 1H, 11α-H), 3.35 (m, 1H, 3-H), 3.15 (m, 1H, 2-H).

Compound 57, i.e.,4′-O-demethyl-4β-[5″-methylthio-2″-(1″,3″,4″-thiadiazolylamino)]-4-desoxypodophyllotoxin.Amorphous, mp 218-220° C. (dec.); ESI MS: 528.3 (M−H); ¹H NMR (300 MHz,CDCl₃+CD₃OD): δ 6.76 (s, 1H, 5-H), 6.55 (s, 1H, 8-H), 6.33 (s, 2H, 2′-H,6′-H), 5.98 (br. d, J=2.2 Hz, 2H, —OCH₂O—), 5.84 (d. J=5.5 Hz, 1H, 4-H),4.68 (d, J=4.9 Hz, 1H, 1-H), 4.44 (t, J=8.2 Hz, 1H, 11β-H), 3.80 (d,J=2.2, 8.8 Hz, 1H, 3-H), 3.78 (s, 6H, 3′,5′-OCH₃), 3.67 (dd, J=4.6, 14.3Hz, 1H, 11α-H), 3.12 (m, 1H, 2-H), 2.87 (s, 3H, SCH₃).

Compound 59, i.e.,4′-O-demethyl-4β-[5″-ethyl-2″-(1″,3″,4″-thiadiazolylamino)]-4-desoxypodophyllotoxin.ESI MS: 510.2 (M−H); ¹H NMR (300 MHz, CDCl₃): δ 6.77 (s, 1H, 5-H), 6.56(s, 1H, 8-H), 6.34 (s, 2H, 2′, 6′-H), 5.97 (d, J=5 Hz, 2H, OCH₂O),5.90(d, J=6 Hz, 1H, 4-H), 4.68 (d, J=5 Hz, 1H, 1-H), 4.42 (t, J=8 Hz,1H, 11-H), 3.78 (s, 6H, 3′, 5′-OCH₃), 3.68-3.83 (m, 2H, 2, 11-H), 3.09(m, 1H, 3-H), 2.56 (q, J=7 Hz, 2, CH ² CH₃), 1.15 (t, J=7 Hz, 3H, CH₂CH₃).

Compound 66, i.e.,4′-O-demethyl-4β-[5″-methylthio-1″H-3″-(1″,2″,4″-triazolylamino)]-4-desoxypodophyllotoxin.Amorphous, mp 195-198° C. (dec.); ESI MS: 511.2 (M−H); ¹H NMR (300 MHz,CDCl₃+CD₃OD): δ 6.60 (d, J=4.4 Hz, 1H, NH of triazole), 6.61 (s, 1H,5-H), 6.59 (s, 1H, 8-H), 6.34 (s, 2H, 2′-H, 6′-H), 5.97 (d, J=16.5 Hz,2H, —OCH₂O—), 5.47 (br. s, 1H, NH on C-4), 5.39 (d, J=5.5 Hz, 1H, 4-H),4.73 (d, J=4.9 Hz, 1H, 1-H), 4.44 (t, J=8.8 Hz, 1H, 11β-H), 4.27 (t,J=7.9 Hz, 1H, 3-H), 3.80 (s, 6H, 3′,5′-OCH₃), 3.45 (dd, J=8.8, 10.4 Hz,1H, 11α-H), 3.05 (m, 1H, 2-H), 2.72 (s, 3H, SCH₃).

Compound 72, i.e.,4′-O-demethyl-4β-(3″,5″-dibromo-2″-pyridylamino)-4-desoxypodophyllotoxin.ESI MS: 633.3 (M−H); ¹H NMR (300 MHz, CDCl₃): δ 8.11 (d, J=2 Hz, 1H,6″-H), 7.81 (d, J=2 Hz, 1H, 4″-H), 6.76 (s, 1H, 5-H), 6.56 (s, 1H, 8-H),6.34 (s, 2H, 2′, 6′-H), 5.99, 5.98 (each d, J=1 Hz, OCH₂O), 5.34 (m, 1H,NH), 5.11 (d, J=6 Hz, 1H, 4-H), 4.63 (d, J=4 Hz, 1H, 1-H), 4.37 (m, 1H,11-H), 3.79 (s, 6H, 3′, 5′-OCH₃), 3.71 (m, 1H, 11-H), 3.03 (m, 2H, 2,3-H).

Compound 73, i.e.,4′-O-demethyl-4β-(1″H-5″-tetrazolylamino)-4-desoxypodophyllotoxin.Amorphous, mp 237-240° C. (dec.); ESI MS: 466.2 (M−H); ¹H NMR (300 MHz,CDCl₃+CD₃OD): δ 6.63 (s, 1H, 5-H), 6.60 (s, 1H, 8-H), 6.35 (s, 2H, 2′-H,6′-H), 6.02 (s, 1H, NH of tetraazole), 5.97 (br. s, 2H, —OCH₂O—), 5.77(d, J=5.5 Hz, 1H, 4-H), 4.77 (d, J=5.5 Hz, 1H, 1-H), 4.37 (t, J=7.9 Hz,1H, 11β-H), 4.11 (t, J=7.1 Hz, 1H, 3-H), 3.78 (s, 6H, 3′,5′-OCH₃), 3.68(dd-like, J=4.9, 13.7 Hz, 1H, 11α-H), 3.30 (m, 1H, 2-H).

Compound 83, i.e.,4′-O-demethyl-4β-(1″-methyl-2″-benzimidazolylamino)-4-desoxypodophyllotoxin.White needles, mp 227-230° C. (dec.); ESI MS: 530 [M+H]. ¹H NMR (CDCl₃)δ 7.49, 7.18 (m, 1H each, H-4″, 7″), 7.14 (m, 2H, H-5″,6″), 6.89 (s, 1H,5-H), 6.57 (s, 1H, 8-H), 6.32 (s, 2H, 2′,6′-H), 6.00 (dd, J=1.1 Hz, 2H,—OCH ² O—), 5.52 (d, J=3.3, 1H, 4-H), 4.65 (d, J=4.4 Hz, 1H, 1-H), 4.51(m, 1H, 11-H), 3.88 (m, 1H, 11-H), 3.79 (s, 6H, 3′,5′-OCH ³ ), 3.52 (s,3H, NCH ³ ), 3.09 (m, 2H, 2-H, 3-H,).

Compound 85, i.e.,4′-O-demethyl-4β-[(1″-methyl-4″-ethoxycarbonyl)-5″-pyrazolylamino]-4-desoxypodophyllotoxin.Amorphous, mp 140-143° C. (dec.); ESI MS: 550.2 (M−H); ¹H NMR (300 MHz,CDCl₃+CD₃OD): δ 7.71 (s, 1H, 3″-H of pyrazole), 6.52 (s, 1H, 5-H), 6.50(s, 1H, 8-H), 6.30 (s, 2H, 2′-H, 6′-H), 5.95 (dd, J=1.1, 7.1 2H, Hz,—OCH₂O—), 5.50 (s, 1H, 4′-OH), 4.92 (d, J=3.8 Hz, 1H, 4-H), 4.89 (d,J=3.8 Hz, 1H, 1-H), 4.64 (d, J=4.9 Hz, 1H, 3-H), 4.37 (dd, J=8.8, 15.9Hz, 1H, 11-H), 4.18 (q, J=7.1 Hz, 2H, OCH ² CH3), 3.78 (s, 6H,3′,5′-OCH₃), 3.74 (s, 1H, N—CH₃), 3.16 (dd, J=4.9, 14.3 Hz, 1H, 11-H),3.05 (m, 1H, 2-H), 1.27 (t, J=7.1 Hz, 3H, OCH2CH ³ .

Compound 88, i.e.,4′-O-demethyl-4β-(3″-amino-5″-pyrazolyloxy)-4-desoxypodophyllotoxin.Amorphous, mp 250-253° C. (dec.); ESI MS: 480.1 (M−H); ¹H NMR (300 MHz,CDCl₃+CD₃OD): δ 6.85 (s, 1H, 5-H), 6.50 (s, 1H, 8-H), 6.30 (s, 2H, 2′-H,6′-H), 5.96 (br. s, 2H, —OCH₂O—), 4.99 (br. s, 1H, 4-H), 4.56 (br. s,1H, 1-H), 4.40 (br. d, 1H, J=8.8 Hz, 11β-H), 4.12 (m, 1H, 3-H), 3.97 (t,1H, J=9.3 Hz, 11α-H), 3.75 (s, 6H, 3′,5′-OCH₃), 3.05 (m, 1H, 2-H).

Compound 90, i.e.,4′-O-demethyl-4β-(1″-benzotriazolyloxy)-4-desoxypodophyllotoxin. ESI MS:516.4 (M−H); ¹H NMR (300 MHz, CDCl₃): δ 8.07 (d, J=9 Hz, 7″-H), 7.56 (m,2H, 5″, 6″-H), 7.44 (s, 1H, 5-H), 6.63 (s, 1H, 8-H), 6.46 (s, 2H, 2′,6′-H), 6.07 (d, J=4 Hz, 2H, OCH₂O), 5.96 (d, J=9 Hz, 1H, 4″-H), 4.65 (d,J=2 Hz, 1H, 1-H, 3.82 (s, 6H, 3′, 5′-OCH₃), 3.77 (m, 1H, 4-H), 3.69,3.61 (each t, J=9 Hz, 11-H), 3.22 (m, 1H, 3-H), 2.88 (dd, J=15, 4 Hz,1H, 2-H).

Compound 91, i.e.,4′-O-demethyl-4β-[3″-(1″,2″,4″-triazolylamino)-4-desoxypodophyllotoxin.Amorphous, mp 245-248° C. (dec.); ESI MS: 465.2 (M−H); ¹H NMR (300 MHz,CDCl₃+CD₃OD): δ 7.74 (br. s, 1H, NH of triazole), 7.38 (s, 1H, 5-H ontriazole), 6.86 (s, 1H, 5-H), 6.53 (s, 1H, 8-H), 6.34 (s, 2H, 2′-H,6′-H), 5.96 (d, J=3.3 Hz, 2H, —OCH₂O—), 5.03 (d, J=3.8 Hz, 1H, 4-H),4.60 (d, J=4.9 Hz, 1H, 1-H), 4.43 (t, J=7.1 Hz, 1H, 11-H), 3.94 (t,J=9.1 Hz, 1H, 11-H), 3.79 (s, 6H, 3′,5′-OCH₃), 3.19 (dd, J=49, 14.2 Hz,1H, 3-H), 3.05 (m, 1H, 2-H).

Compound 94, i.e.,4′-O-demethyl-4β-(3″-quinolinylamino)-4-desoxypodophyllotoxin. ESI MS:525.3 (M−H); ¹H NMR (300 MHz, CDCl₃): δ 8.43 (d, J=3 Hz, 1H, 2″-H), 7.96(m, 1H, 8″-H), 7.63 (m, 1H, 5″-H), 7.47 (m, 2H, 6″, 7″-H), 6.99 (d, J=3Hz, 1H, 4″-H), 6.77 (s, 1H, 5-H), 6.54 (s, 1H, 8-H), 6.35 (s, 2H, 2′,6′-H), 5.96 (d, J=7 Hz, OCH₂O), 5.70 (br s, 1H, 4′-OH), 4.78 (m, 1H,NH), 4.60 (d, J=4 Hz, 1H, 4-H), 4.46 (d, J=6 Hz, 1H, 1-H), 4.44 (m, 1H,11-H), 3.96 (t, J=9 Hz, 1H, 11-H), 3.79 (s, 6H, 3′, 5′-OCH3), 3.14 (m,2H, 2, 3-H).

Compound 98, i.e.,4′-O-demethyl-4β-[5″-(3″-methyl)-isoxazolylamino]-4-desoxypodophyllotoxin.Amorphous, mp 227-229° C. (dec.); ESI MS: 479.1 (M−H); ¹H NMR δ (Bruker400 MHz, CDCl₃): 6.80 (s, 1H, 5-H), 6.54 (s, 1H, 8-H), 6.30 (s, 2H,2′-H, 6′-H), 5.98 (dd, 2H, J=1.2, 9.0 Hz, —OCH₂O—), 5.43 (s, 1H, 4″-H ofisoxazole), 4.74 (d, J=3.5, 1H, 4-H), 4.60 (d, J=4.3 Hz, 1H, 1-H), 4.40(dd, J=7.0, 8.2 Hz, 1H, 11β-H), 4.00 (t, J=9.0 Hz, 1H, 2-H), 3.79 (s,6H, 3′,5′-OCH₃), 3.03 (dd, J=4.7, 1H, 14.1 Hz, 11α-H), 2.98 (1H, m,3-H), 2.20 (3H, s, CH₃ of isoxazole).

Compound 105, i.e.,4′-O-demethyl-4β-[2″-(5″-methyl)-benzothiazolylamino]-4-desoxypodophyllotoxin.Amorphous, mp 245-248° C. (dec.); ESI MS: 545.2 (M−1); ¹H NMR δ (Bruker400 MHz, CDCl₃): 7.45 (1H, d, J=8.2 Hz, 4″-H), 7.41 (1H, br. s, 7″-H),7.15 (1H, br. d, J=8.2 Hz, 5″-H), 6.89, 6.54 (1H each, both s, 5-H,8-H). 6.32 (2H, s, 2′-H, 6′-H), 5.98 (2H, dd, J=1.2, 7.8 Hz, —OCH₂O—),5.42 (1H, d, J=3.9 Hz, 4-H), 4.61 (1H, d, J=4.3 Hz, 1-H), 4.50 (1H, dd,J=6.7, 9.0 Hz, 2-H), 3.98 (1H, dd, J=9.8, 20.0 Hz, 11α-H), 3.79 (6H, s,3′,5′-OCH₃), 3.08 (1H, m, 3-H), 3.01 (1H, dd, J=4.3, 13.7 Hz, 11β-H),2.42 (3H, s, CH₃).

Compound 106, i.e.,4′-O-demethyl-4β-[3″-(5″-nitro)-benzisothiazolylamino]-4-desoxypodophyllotoxin.Amorphous, mp 114-117° C.; ESI MS: 576.3 (M−H); ¹H NMR (Bruker 400 MHz,CDCl₃): δ 8.72 (s, 1H, 4″-H), 8.09 (dd, J=2.3, 12.1 Hz, 1H, 6″-H), 7.51(d, J=9.8 Hz, 1H, 7″-H), 6.77 (s, 1H, 5-H), 6.62 (s, 1H, 8-H), 6.35 (s,2H, 2′, 6′-H), 5.99 (d, J=1.2 Hz, 2H, OCH₂O), 5.46 (br s, 1H, 4′-OH),4.73 (d, J=4.7 Hz, 1H, 1-H), 4.46 (m, 1H, 4-H), 4.48 (t, J=7.0 Hz, 1H,11-H), 3.95 (t, J=10.1 Hz, 1H, 11-H), 3.81 (s, 6H, 3′, 5′-OCH3), 3.22(m, 2H, 2, 3-H).

Compound 110, i.e.,4′-O-demethyl-4β-[2″-(5″-trifluroumethyl-1″,3″,4″-thiadiazolylamino)]-4-desoxypodophyllotoxin.Amorphous, mp 225-227° C. (dec.); ESI MS:550.4 (M−H); ¹H NMR (Bruker 400MHz, CDCl₃): δ 6.70 (1H, s, 5-H), 6.57 (1H, s, 8-H), 6.32 (2H, s, 2′-H,6′-H), 6.00, (2H, dd, J=1.2, 4.3 Hz, —OCH₂O—), 5.97 (1H, d, J=5.5 Hz,4-H), 4.71 (1H, d, J=4.7 Hz, 1-H), 4.42 (1H, dd, J=7.4, 8.8 Hz, 11β-H),3.79 (6H, s, 3′,5′-OCH₃), 3.70 (1H, dd, J=9.0, 11.0 Hz, 11α-H), 3.47(1H, dd, J=4.7, 5.1 Hz, 2-H), 3.14 (1H, m, 3-H).

Compound 144, i.e.,4′-O-demethyl-4β-[4″-(2″,1″,3″-benzothiadiazolylamino)]-desoxypodophyllotoxin.Amorphous, mp 168-172° C.; ESI MS: 532.0 (M−H); ¹H NMR (Bruker 400 MHz,CDCl₃): δ 7.48 (m, 1H, 6″-H), 7.36 (d, J=8.6 Hz, 1H, 7″-H), 6.77 (s, 1H,5-H), 6.58 (s, 1H, 8-H), 6.36 (s, 2H, 2′, 6′-H), 6.34 (d, J=7.8 Hz, 1H,1H, 5″-H), 5.98 (dd, J=1.2, 8.6 Hz, 2H, OCH ² O), 5.43 (br s, 1H,4′-OH), 4.88 (t, J=4.3 Hz, 1H, 4-H), 4.46 (d, J=4.7 Hz, 1H, 1-H), 4.44(t, J=8.2 Hz, 1H, 11-H), 3.98 (t, J=10.6 Hz, 1H, 11-H), 3.81 (s, 6H, 3′,5′-OCH3), 3.24 (dd, J=4.7 Hz, 1H, 2-H), 3.13 (m, 1H, 3-H).

Compound 155, i.e.,4′-O-demethyl-4β-[2″-(5″-chlorobenzoxazolylamino)]-desoxypodophyllotoxin.Amorphous, mp 215-217° C. (dec.); ESI MS: 549.2 (M−H); ¹H NMR (Bruker400 MHz, CDCl₃): δ 6.95 (1H, d, J=8.2 Hz, 7″-H), 6.88 (1H, dd, J=2.0,8.2 Hz, 6″-H), 6.72, 6.71 (1H each, both s, 5-H, 8-H). 6.37 (2H, s,2′-H, 6′-H), 6.02 (2H, dd, J=1.2, 7.8 Hz, —OCH₂O—), 5.71 (1H, d, J=2.0Hz, 4″-H), 5.67 (1H, d, J=5.1 Hz, 4-H), 4.81 (1H, d, J=5.1 Hz, 1-H),4.58 (1H, dd, J=7.0, 9.4 Hz, 2-H), 3.87 (1H, dd, J=10.6, 18.8 Hz,11α-H), 3.80 (6H, s, 3′,5′-OCH₃), 3.18 (1H, m, 3-H), 3.01 (1H, dd,J=5.1, 14.5 Hz, 11β-H).

Compound 160, i.e.,4′-O-demethyl-4β-[2″-(5″-chloropyridinlyloxy)]-desoxypodophyllotoxin.Amorphous, mp 205-208° C. (dec); ESI MS: 521.2 (M−H); ¹H NMR (Bruker 400MHz, CDCl₃): δ 8.16 (1H, dd, J=3.1, 9.8 Hz, 4″-H of pyridine), 8.02 (1H,d, J=3.1 Hz, 6″-H of pyridine), 6.70 (1H, s, 5-H), 6.67 (1H, d, J=9.8Hz, 3″-H of pyridine), 6.49 (1H, s, 8-H), 6.32 (2H, s, 2′-H, 6′-H), 6.24(1H, d, J=5.1 Hz, 4-H), 6.06 (2H, br. dd, J=1.2, 2.3 Hz, —OCH₂O—), 4.79(1H, d, J=5.1 Hz, 1-H), 4.48 (1H, dd, J=7.0, 9.4 Hz, 2-H), 3.81 (6H, s,3′,5′-OCH₃), 3.38 (1H, dd, J=9.4, 11.0 Hz, 11α-H), 3.28 (1H, m, 3-H),2.75 (1H, dd, J=5.1 14.1 Hz, 11β-H).

Compound 188, i.e.,4′-O-demethyl-4β-[6″-(2″-mercaptobenzothiazolylamino)]-desoxypodophyllotoxin.Colorless needles, mp 219-221° C. (dec.); ESI MS: 563.4 (M−H); ¹H NMR(Bruker 400 MHz, CDCl₃): δ 7.62 (1H, d, J=8.6 Hz, 4″-H ofbenzothiazole), 7.03 (1H, d, J=2.0 Hz, 7″-H of benzothiazole), 7.01 (1H,s, 5-H), 6.79 (1H, dd, J=2.0, 8.6 Hz, 5″-H of benzothiazole), 6.49 (1H,s, 8-H), 6.33 (2H, s, 2′-H, 6′-H), 5.97 (2H, dd, J=1.2, 9.4 Hz,—OCH₂O—), 5.66 (1H, d, J=3.9 Hz, 4-H), 4.60 (1H, d, J=5.1 Hz, 1-H), 4.42(1H, dd, J=7.4, 9.0 Hz, 2-H), 4.01 (1H, dd, J=9.0, 10.6 Hz, 11α-H), 3.80(6H, s, 3′,5′-OCH₃), 3.33 (1H, m, 3-H)), 3.20 (1H, dd, J=8.6, 13.7 Hz,11β-H).

Synthesis of Compounds 102 and 135

Each of these two compounds was synthesized as followed. Reaction of anamino substituted heteroaryl carboxylate with a reducing reagent, e.g.,lithium aluminum hydride (1.3-2.0 eq), in a solvent of ether andtetrohydrofuran (3:1) yielded an alcohol as shown in Scheme 5. Theresulting alcohol then reacted with DBD to give the desired compound.

Analytical data on Compound 102 are shown below.

Compound 102, i.e., 4′-O-demethyl-4β-[2″-(4″-hydroxylethyl)-thiazolylamino]-desoxypodophyllotoxin. Amorphous,mp 128° C. (soften) 155-8° C. (dec); ESI MS: 526.2 (M−H); ¹H NMR (Bruker400 MHz, CD₃OD): δ 6.82 (s, 1H, 5-H), 6.49 (s, 1H, 8-H), 6.32 (s, 2H,2′, 6′-H), 6.24 (s, 1H, 5″-H), 5.92 (d, J=2.0 Hz, 2H, OCH₂O), 5.20 (d,J=4.3 Hz, 1H, 4-H), 4.56 (d, J=5.1 Hz, 1H, 1-H), 4.39 (t, J=7.4 Hz, 1H,11-H), 3.89 (t, J=8.9 Hz, 1H, 11-H), 3.80 (t, J=6.6 Hz, 2H, —CH₂ CH ²OH), 3.71 (s, 6H, 3′, 5′-OCH3), 3.15 (dd, J=5.1, 14.5 Hz, 1H, 2-H), 3.04(m, 1H, 3-H), 2.74 (t, J=6.6 Hz, 2H, —CH ² CH₂OH).

Synthesis of Compounds 107 141, 142, and 148

Each of Compounds 107 and 148 was synthesized as shown in Scheme 6. Achloro and nitro substituted heteroaryl was treated with a substitutedamine (2 eq) in carbon tetrachloride. The reaction solution was heatedto reflux for 24 h. The resulting compound as yellow crystal was furtherrefluxed in a mixture of methanol and water with 10% glacial acetic acidfor 1 to 2 h in the presence of iron powder to give an amino substitutedheteroaryl intermediate. Reaction of the intermediate with DBD yieldedthe desired product.

Each of Compounds 141 and 142 was synthesized as follows. A chloro andnitro substituted heteroaryl reacted with an alcohol in the presence ofsodium to give an ether. The nitro group of the resulting ether wasreduced with iron powder to give an amine compound which reacted withDBD to yield the desired product.

Analytical data on Compound 107 are shown below.

Compound 107, i.e.,4′-O-demethyl-4β-[4″-(2″-N,N-diethyl)-pyridylamino]-desoxypodophyllotoxin.Amorphous, mp 92° C. (shrunken) 140-142° C. (melted); ESI MS: 546.4(M−H); ¹H NMR (Bruker 400 MHz, CDCl₃): δ 7.61 (s, 1H, 6″-H), 6.71 (s,1H, 4″-H), 6.52 (s, 2H, 3″-H, 5-H), 6.39 (s, 1H, 8-H), 6.34 (s, 2H, 2′,6′-H), 5.96 (dd, J=1.2, 6.7 Hz, 2H, OCH₂O), 4.59 (d, J=5.1 Hz, 1H, 1-H),4.51 (t, J=7.4 Hz, 1H, 11-H), 4.42 (t, J=8.6 Hz, 1H, 4-H), 4.05 (t,J=8.9 Hz, 1H, 11-H), 3.80 (s, 6H, 3′, 5′-OCH3), 3.46 (m, 4H, —N(CH ²CH₃)₂), 3.16 (m, 1H, 2-H), 2.99 (m, 1H, 3-H), 1.18 (m, 6H, —N(CH₂ CH ³)₂).

Synthesis of Compounds 159, 179, 189, 190, and 191

Compound 159 was synthesized as shown in Scheme 7. Reaction of5-amino-3-methyl isothiazole hydrochloride (2 mmol) with bromine (2mmol) in a solution of 5% glacial acetic acid in benzene (5 mL) at 10°C. provided a solid product as a hydrobromide salt. The solid productwas converted to a free base product by stirring with 2N sodiumcarbonate (D. Buttimore et al. (1963) JACS 2032-2039). Reaction theresulting compound with DBD under N₂ with refluxing yielded the desiredproduct.

Compounds 179 and 189 were synthesized as shown Scheme 7. Reaction of5-amino-3-methylisothiazole hydrochloride with acetic chloride inpyridine followed by nitration and reduction gave an intermediate of4-amino-5-acetamido-3-methylisothiazole. Reaction of the intermediate(1.2 eq ) with DBD under N₂ afforded Compound179. Refluxing4-nitro-5-acetamido-3-methylisothiazole in 4N HCl aq. and furtherreacting with DBD under N₂ with refluxing yielded Compound 189.

Each of Compounds 190 and 191 was synthesized as shown in Scheme 7.Reaction of 5-amino-3-methylisothiazole hydrochloride with a substitutedacetic chloride (2 eq) in pyridine at room temperature, followed bynitration of the resulting compound,5-dichloroacetamido-3-methylisothiazole, in fuming nitric acid (1.1 eq)and concentrated sulfuric acid at 0° C. gave a nitro compound. Reductionof the nitro compound with iron powder afforded an amino compound.Reaction of the resulting amino compound with DBD afforded the desiredproduct.

Analytical data on Compound 190 are shown below.

Compound 190, i.e.,4′-O-demethyl-4β-[4″-(5″-chloroacetamido-3″-methyl)-isothiazolylamino]-desoxypodophyllotoxin.Amorphous, mp 178-180° C. (dec); ESI MS: 586.2 (M−H); ¹H NMR (Bruker 400MHz, CD₃OD): δ 6.54 (s, 1H, 5-H), 6.25 (s, 2H, 2′, 6′-H), 6.12 (d, J=5.9Hz, 1H, 8-H), 5.91 (m, 2H, OCH ² O), 4.68 (d, J=5.5 Hz, 1H, 1H, 1-H),4.63 (m, 1H, 4-H), 4.47 (t, J=8.2 Hz, 1H, 11-H), 4.21 (t, J=3.9 Hz, 1H,11-H), 3.76 (s, 6H, 3′, 5′-OCH3), 3.45 (m, 2H, —CH ² Cl), 3.40 (m, 1H,2-H), 3.08 (m, 1H, 3-H), 2.31 (s, 3H, 3″-H).

Synthesis of Compounds 200 and 201

Compound 200 was synthesized as shown in Scheme 8. Reaction ofα,γ-dichloroacetone (15.9 mmol) with thiourea (15.9 mmol) in dry acetone(8 mL) afforded a white solid. The white solid was collected and stirredin anhydrous ethanol to remove insoluble isothiourea. To the ethanolfiltrate, 25-30 mL of hexanes was added with stirring to afford2-amino-4-chloromethylthiazole hydrochloride as a white crystallinesolid. The resulting compound was further reacted with N,N-diethylaminein ethanol and neutralized with 20% sodium hydroxide to gave anintermediate. The intermediate reacted with DBD to yield the desiredproduct.

Compound 201, i.e.,4′-O-demethyl-4β-[2″-(4″-hydroxylmethyl)-thiazolylamino]-desoxypodophyllotoxinwas synthesized as follows. 2-amino-4-chloromethylthiazole hydrochloride(42.0 mmol) in 16 mL of water was heated to reflux for 15 min. Therection solution was evaporated to dryness and the residue wasredissolved in water and evaporated. The residue was crystallized fromethanol to give an alcohol intermediate. The intermediate furtherreacted with DBD under reflux condition to afforded the title compoundin 35% yield. Amorphous, mp 185-189° C.; ESI MS: 511.2 (M−H); ¹H NMR(Bruker 400 MHz, CD₃OD): δ 6.82 (s, 1H, 5-H), 6.49 (s, 1H, 8-H), 6.43(s, 1H, 5″-H), 6.32 (s, 2H, 2′, 6′-H), 5.92 (d, J=2.7 Hz, 2H, OCH₂O),5.24 (d, J=4.3 Hz, 1H, 4-H), 4.56 (d, J=5.1 Hz, 1H, 1-H). 4.45 (s, 2H,—CH ² OH), 4.41 (t, J=7.4 Hz, 1H, 11-H), 3.88 (t, J=8.9 Hz, 1H, 11-H),3.71 (s, 6H, 3′, 5′-OCH3), 3.15 (dd, J=5.1, 14.5 Hz, 1H, 2-H), 3.04 (m,1H, 3-H).

Synthesis of Compounds 207-210

Each of these compounds was synthesized as follows. Reaction of thepodophyllotoxin derivatives was treated with phosphorous oxychloride (2eq for the compounds 207-209, and 4 eq for the compound 210) in thepresence of N,N-diisopropylethylamine (5 eq and 10 eq, respectively) at−20 to −15° C. The resulting product was hydrolyzed in water at −5 to 0°C. in the presence of pyridine to provide the desired product.

Analytical data on Compound 210 are shown below.

Compound 210, i.e.,4′-O-demethyl-4β-[2″-(4″-hydroxylethyl)-thiazolylamino]-desoxypodophyllotoxin-4′-O,4″O-diphosphate.Amorphous, mp 176° C. (dec); ESI MS: 685.2 (M−H); ¹H NMR (Bruker 400MHz, DMSO-d₆): δ 6.88 (s, 1H, 5-H), 6.52 (s, 1H, 8-H), 6.32 (s, 1H,5″-H), 6.22 (s, 2H, 2′, 6′-H), 5.98 (d, J=4.70 Hz, 2H, OCH₂O), 5.28 (d,J=4.7 Hz, 1H, 4-H), 4.52 (d, J=5.1 Hz, 1H, 1-H), 4.34 (t, J=7.4 Hz, 1H,11-H), 3.92 (m, 2H, —CH₂ CH ² OPO₃H₂), 3.78 (t, J=8.6 Hz, 1H, 11-H),3.58 (s, 6H, 3′, 5′-OCH₃), 2.72 (m, 1H, 2-H), 2.67 (m, 1H, 3-H), 2.33(t, J=2.0 Hz, 2H, —CH ² CH₂O PO₃H₂).

Biological Assays

A number of compounds of this invention were evaluated for cytotoxicityagainst KB cells, which are nasopharyngeal carcinoma cells. They werealso tested for stimulation of cellular protein-linked DNA breaks (PLDB)using etoposide as a positive control. Etopside is a widely-usedantineoplastic agent, see, e.g., Zhang et al. (1994) J. Med. Chem. 37:446.

Among the tested compounds, Compounds 1, 15, 36, 39, 45, and 49 showedunexpectedly low IC₅₀ values against KB cells and are therefore strongcytotoxic agents against cancer cells. Indeed, Compounds 1, 36, 39 and49 showed unexpectedly high levels of PLDB induction in KB cells whentested at 5 μg/ml.

Three compounds, i.e., Compounds 1, 12, and 38, were also assayed forinhibition of tubulin polymerization in vitro. The results showed thatnone of them inhibited tubulin polymerization at concentrations as highas 40 μM, indicating that the growth inhibitory activity of thesecompounds did not result from inhibition of tubulin polymerization.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. For example, compounds structurally analogouspodophyllotoxin derivatives of this invention also can be made, screenedfor their anticancer activities, and used to practice this invention.Thus, other embodiments are also within the claims.

1. A compound of formula (I):

wherein each of R₁, R₂, R₃ and R₇ independently is H or alkyl; each ofR₄ and R₆ independently is alkyl; R₅ is H or P(O)(OR_(a))₂, in whichR_(a) is H or alkyl; T is H, or together with X is ═N; X is a bond, O,S, or NR_(b), in which R_(b) is H or alkyl; or together with T, is ═N;and Y is 5-membered heteroaryl or heterocyclyl, optionally substitutedwith one or more of halogen, alkyl, cyclyl, aryl, heteroaryl,heterocyclyl, —OR_(c), —NR_(c)R_(c)′, —SR_(c), —CN, —NO₂, —SO₂R_(c),—C(O)OR_(c), —C(O)NR_(c)R_(c)′, —NHC(O)R_(c), —(CH₂)_(q)OPO₃H₂,—CH₂C(O)NOR_(c)″, and

 in which each of R_(c) and R_(c)′ independently is H or alkyl; Rc″ isH, alkyl, or silyl; Z is O or NH; each of m and n independently is 0 or1; p is 0, 1, or 2; q is 1, 2, 3, or 4; and each of R₈ and R₉independently is H, alkyl, aryl, heteroaryl, heterocyclyl, —OR_(d),—NR_(d)R_(d)′, —SR_(d), —CN, —NO₂, —SO₂R_(d), —C(O)OR_(d),—C(O)NR_(d)R_(d)′, —NHC(O)R_(d), or —NHC(O)OR_(d), in which each ofR_(d) and R_(d)′ independently is H or alkyl.
 2. The compound of claim1, wherein X is NH, and T is H.
 3. The compound of claim 2, wherein eachof R₁, R₂, R₃, and R₇ is H.
 4. The compound of claim 3, wherein R₅ is H.5. The compound of claim 3, wherein R₅ is P(O)(OH)₂.
 6. The compound ofclaim 3, wherein each of R₄ and R₆ is methyl.
 7. The compound of claim6, wherein R₅ is H.
 8. The compound of claim 7, wherein Y is 5-memberedheteroaryl.
 9. The compound of claim 8, wherein Y is


10. The compound of claim 8, wherein Y is 5-membered heteroarylcontaining two to four ring heteroatoms.
 11. The compound of claim 10,wherein Y is


12. The compound of claim 10, wherein Y is


13. The compound of claim 10, wherein Y is


14. The compound of claim 10, wherein Y is


15. The compound of claim 10, wherein Y is


16. The compound of claim 10, wherein Y is


17. The compound of claim 10 wherein Y is


18. The compound of claim 17, wherein m is
 1. 19. The compound of claim18, wherein n is
 0. 20. The compound of claim 19, wherein Z is O. 21.The compound of claim 18, wherein n is
 1. 22. The compound of claim 21,wherein R₉ is C(O)OR_(d).
 23. The compound of claim 22, wherein Z is O.24. The compound of claim 17, wherein m is
 0. 25. The compound of claim7, wherein Y is 5-membered heterocyclyl.
 26. The compound of claim 2,wherein each of R₄ and R₆ is methyl.
 27. The compound of claim 1,wherein X and T together are ═N.
 28. The compound of claim 27, whereineach of R₁, R₂, R₃, and R₇ is H.
 29. The compound of claim 28, whereineach of R₄and R₆ is methyl.
 30. The compound of claim 29, wherein R₅ isH.
 31. The compound of claim 28, wherein R₅ is H.
 32. The compound ofclaim 27, wherein each of R₄ and R₆ is methyl.
 33. A method for treatingcancer, comprising administering to a subject in need thereof aneffective amount of a compound of formula (I):

wherein each of R₁, R₂, R₃ and R₇ independently is H or alkyl; each ofR₄ and R₆ independently is alkyl; R₅ is H or P(O)(OR_(a))₂, in whichR_(a) is H or alkyl; T is H, or together with X is ═N; X is a bond, O,S, or NR_(b), in which R_(b) is H or alkyl; or together with T, is ═N;and Y is 5-membered heteroaryl or heterocyclyl, optionally substitutedwith one or more of halogen, alkyl, cyclyl, aryl, heteroaryl,heterocyclyl, —OR_(c), —NR_(c)R_(c)′, —SR_(c), —CN, —NO₂, —SO₂R_(c),—C(O)OR_(c), —C(O)NR_(c)R_(c)′, —NHC(O)R_(c), —(CH₂)_(q)OPO₃H₂,—CH₂C(O)NOR_(c)″, and

 in which each of R_(c) and R_(c)′ independently is H or alkyl; R_(c)″is H, alkyl, or silyl; Z is O or NH; each of m and n independently is 0or 1; p is 0, 1, or 2; q is 1, 2, 3, or 4; and each of R₈ and R₉independently is H, alkyl, aryl, heteroaryl, heterocyclyl, —OR_(d),—NR_(d)R_(d)′, —SR_(d), —CN, —NO₂, —SO₂R_(d)—C(O)OR_(d),—C(O)NR_(d)R_(d)′, —NHC(O)R_(d), or —NHC(O)OR_(d), in which each ofR_(d) and R_(d)′ independently is H or alkyl.
 34. The method of claim33, wherein X is NH, and T is H.
 35. The compound of claim 34, whereineach of R₄ and R₆ is methyl.
 36. The compound of claim 34, wherein eachof R₁, R₂, R₃, and R₇ is H.
 37. The compound of claim 36, wherein R₅ isH.
 38. The compound of claim 36, wherein R₅ is P(O)(OH)₂.
 39. Thecompound of claim 36, wherein each of R₄ and R₆ is methyl.
 40. Thecompound of claim 39, wherein R₅ is H.
 41. The compound of claim 40,wherein Y is 5-membered heteroaryl.
 42. The compound of claim 41,wherein Y is


43. The compound of claim 41, wherein Y is 5-membered heteroarylcontaining two to four ring heteroatoms.
 44. The method of claim 43,wherein Y is